Expression of recombinant ribosome inactivating protein MAP30 in E.coli and its biological activity / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.</p><p><b>METHODS</b>The DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.</p><p><b>RESULT</b>The nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.</p><p><b>CONCLUSION</b>The gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.</p>